Cyclophasphamide is the most widely used agent in the treatment of numerous hematologic and solid human tumors. The long-term objective of this research is to develop new types of cyclophosphamide-based antitumor agents and study their mechanisms of activation, action, and toxicity. The approach will be based upon the specific chemical mechanistic information discovered during the previous grant period. This information will be utilized to guide the design, synthesis, and evaluation of new mechanism-based phosphoramidate antitumor agents. Specific aims include 1) Determination of the cytotoxic contribution of intracellular vs. extracellular release of the alkylating agent phosphoramide mustard; 2) Continuation of the synthesis, development, and cytotoxic evaluation of substituted aldophosphamide analogs designed for activity against cyclophosphamide-resistant cells; 3) Development of a new series of cyclophosphamide analogs designed as bioreductive alkylating agents to achieve cytotoxic specificity against hypoxic cells; 4) Development of a series of aldophosphamide analogs designed to deliver cytotoxic nucleotides and analogs to tumor cells. Nuclear magnetic resonance (NMR) will be the primary technique used to measure drug activation and breakdown in vitro, and this data will be used in the design of new analogs. All new compounds will initially be evaluated in cell culture against established murine tumor lines, and compounds showing activity will also be tested in vivo. Our goal is to utilize the unique activation pathways of cyclophosphamide to develop new drugs with reduced host toxicity and/or increased efficacy against resistant or poorly responsive tumor cells.